1. Field of the Invention
The present invention relates to amplifying sequences and vectors comprising these sequences, to the use of these vectors in compositions for the expression of nucleotide sequences in these transfected cells, and to the use of these products for therapeutic and vaccine applications.
2. Description of the Background
The desmine gene, one of the first muscle proteins to be detected in the developing embryo of mammals, was sequenced in 1989 by Li e. al. (Gene, 78,243-254).
Subsequently, an amplifying sequence of 280 base pairs, situated between the nucleotides -693 and -973 upstream from the transcription initiation site, was described (Li and Paulin, 1991, J.Biol.Chem., 266 6562-6570).
This amplifying sequence was identified by producing a series of plasmid constructions carrying the bacterial gene coding for chloramphenicol acetyl transferase (CAT), and by introducing them into myogenic or non-myogenic mice cells. The authors show that this amplifying region of 280 base pairs can activate both homologous and heterologous promoters regardless of their orientation, position or distance in relation to this sequence.
The study of this sequence was continued (Li et al., 1993,J.Biol.Chem., 268,10 403-10.415) and showed that this sequence contained two regions: one active in myotubes and the other active in myoblasts.
The part that is active in myotubes lies between positions -973 and -848, that is to say in a sequence of 125 nucleotides.
Within this sequence, a region lying between positions -910 and -870 is protected from the action of a DNase. This region contains binding sites of MEF2 and MyoD1 factors. No specific activity is indicated for this region of 40 nucleotides. In particular, no experiment has been carried out with constructions containing this isolated region. Mention has only been made that MyoD1 and MEF2 sites are necessary to obtain full amplifying activity in the myotubes.
The amplifying effect of the sequence upstream from the desmine gene has also been tested in a transgenic mouse (Li et al., 1993, Development, 117,947-959). A fragment of 1 kb containing the regulatory sequence upstream from the desmine gene is bound to a reporting gene coding for Escherichia coli .beta.-galactosidase. The authors show that the amplifying activity of the desmine promoter is very considerable as the activity of .beta.-galactosidase can be easily detected in tissue sections.
It arises from the prior art analysed above, that the amplifying properties of certain parts upstream from the human desmine gene are known. On the other hand, the regions responsible for this amplifying activity have not been identified.
They could therefore not be successfully modified with a view to improving their performance for the expression of genes or nucleotide sequences corresponding in whole or in part to these c-DNAs (complementary DNAs) coding for proteins of interest or for fragments of these proteins conveying epitopes able to induce protective antibodies or antibodies which recognise said epitopes. The preferable length of c-DNA or genomic DNA fragments is between 30 base pairs and 2 kb. They can be produced by chemical synthesis or by restriction enzyme cutting of DNA extracted from cells or micro-organisms selected by methods known to men of the art.
It is known that by introducing DNA into these eucaryote cells using the described methods, in particular by patent WO-90 11 092, it is not possible to obtain an extended or a high quality expression of said DNAs introduced in plasmid form.